Phylogenetic analysis of the fecal flora of the wild pygmy loris

The bacterial diversity in fecal samples from the wild pygmy loris was examined with a 16S rDNA clone library and restriction fragment length polymorphism analysis. The clones were classified as Firmicutes (43.1%), Proteobacteria (34.5%), Actinobacteria (5.2%), and Bacteroidetes (17.2%). The 58 different kinds of 16S rDNA sequences were classified into 16 genera and 20 uncultured bacteria. According to phylogenetic analysis, the major genera within the Proteobacteria was Pseudomonas, comprising 13.79% of the analyzed clone sequences. Many of the isolated rDNA sequences did not correspond to known microorganisms, but had high homology to uncultured clones found in human feces. Am. J. Primatol. 72:699–706, 2010. © 2010 Wiley‐Liss, Inc.     

山西省草兔的一些生态资料

山西省草兔的一些生态资料卢欣，申守义，高尚文（山西省生物研究所太原０３０００６）关键词草兔，粪便样方，益害分析，天敌和寄生虫草兔（Ｌｅｐｕｓｃａｐｅｎｓｉｓ）是我国最重要的小型狩猎动物之一，但其生态仅有零星报道［１，２］。１９８７－１９９２年，我们在...     

Determination of the functional epitopes of human interleukin-18-binding protein by site-directed mutagenesis

The human interleukin (IL)-18-binding protein (hIL-18BP) is a naturally occurring antagonist of IL-18, a proinflammatory cytokine that is related to IL-1beta and has an important role in defense against microbial invaders. As its name implies, the hIL-18BP binds to IL-18 with high affinity and prevents the interaction of IL-18 with its receptor. We genetically modified the C terminus of hIL-18BP by appending a 15-amino acid biotinylation recognition site and a six-histidine tag and then performed site-directed mutagenesis to determine the functional epitopes that mediate efficient binding to IL-18. The mutated IL-18BPs were secreted from mammalian cells, captured by metal affinity chromatography, biotinylated in situ, eluted, and immobilized on streptavidin-coated chips. Using surface plasmon resonance, we identified seven amino acids of hIL-18BP which, when changed individually to alanine, caused an 8-750-fold decrease in binding affinity, largely because of increased off-rates. These seven amino acids localized to the predicted beta-strand c and d of hIL-18BP immunoglobulin-like domain, and most had hydrophobic side chains. Just two amino acids, tyrosine 97 and phenylalanine 104, contributed approximately 50% of the binding free energy. Information obtained from these studies could contribute to the design of molecular antagonists of IL-18 for treatment of inflammatory diseases.     

Identification, substrate specificity, and inhibition of the Streptococcus pneumoniae beta-ketoacyl-acyl carrier protein synthase III (FabH)

In the bacterial type II fatty acid synthase system, beta-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) catalyzes the condensation of acetyl-CoA with malonyl-ACP. We have identified, expressed, and characterized the Streptococcus pneumoniae homologue of Escherichia coli FabH. S. pneumoniae FabH is approximately 41, 39, and 38% identical in amino acid sequence to Bacillus subtilis, E. coli, and Hemophilus influenzae FabH, respectively. The His-Asn-Cys catalytic triad present in other FabH molecules is conserved in S. pneumoniae FabH. The apparent K(m) values for acetyl-CoA and malonyl-ACP were determined to be 40.3 and 18.6 microm, respectively. Purified S. pneumoniae FabH preferentially utilized straight short-chain CoA primers. Similar to E. coli FabH, S. pneumoniae FabH was weakly inhibited by thiolactomycin. In contrast, inhibition of S. pneumoniae FabH by the newly developed compound SB418011 was very potent, with an IC(50) value of 0.016 microm. SB418011 also inhibited E. coli and H. influenzae FabH with IC(50) values of 1.2 and 0.59 microm, respectively. The availability of purified and characterized S. pneumoniae FabH will greatly aid in structural studies of this class of essential bacterial enzymes and facilitate the identification of small molecule inhibitors of type II fatty acid synthase with the potential to be novel and potent antibacterial agents active against pathogenic bacteria.     

A Transmission/Disequilibrium Test That Allows for Genotyping Errors in the Analysis of Single-Nucleotide Polymorphism Data

The present study assesses the effects of genotyping errors on the type I error rate of a particular transmission/disequilibrium test (TDT(std)), which assumes that data are errorless, and introduces a new transmission/disequilibrium test (TDT(ae)) that allows for random genotyping errors. We evaluate the type I error rate and power of the TDT(ae) under a variety of simulations and perform a power comparison between the TDT(std) and the TDT(ae), for errorless data. Both the TDT(std) and the TDT(ae) statistics are computed as two times a log-likelihood difference, and both are asymptotically distributed as chi(2) with 1 df. Genotype data for trios are simulated under a null hypothesis and under an alternative (power) hypothesis. For each simulation, errors are introduced randomly via a computer algorithm with different probabilities (called "allelic error rates"). The TDT(std) statistic is computed on all trios that show Mendelian consistency, whereas the TDT(ae) statistic is computed on all trios. The results indicate that TDT(std) shows a significant increase in type I error when applied to data in which inconsistent trios are removed. This type I error increases both with an increase in sample size and with an increase in the allelic error rates. TDT(ae) always maintains correct type I error rates for the simulations considered. Factors affecting the power of the TDT(ae) are discussed. Finally, the power of TDT(std) is at least that of TDT(ae) for simulations with errorless data. Because data are rarely error free, we recommend that researchers use methods, such as the TDT(ae), that allow for errors in genotype data.     

DNA array and biological characterization of the impact of the maturation status of mouse dendritic cells on their phenotype and antitumor vaccination efficacy

We systematically investigated the impact of the relative maturation levels of dendritic cells (DCs) on their cell surface phenotype, expression of cytokines and chemokines/chemokine receptors (by DNA array and RNase protection analyses), biological activities, and abilities to induce tumor immunity. Mature DCs expressed significantly heightened levels of their antigen-presenting machinery (e.g., CD54, CD80, CD86) and numerous cytokines and chemokines/chemokine receptors (i.e., Flt-3L, G-CSF, IL-1alpha and -1beta, IL-6, IL-12, CCL-2, -3, -4, -5, -17, and -22, MIP-2, and CCR7) and were significantly better at inducing effector T cell responses in vitro. Furthermore, mice vaccinated with tumor peptide-pulsed mature DCs better survived challenge with a weakly immunogenic tumor (8 of 8 survivors) than did mice vaccinated with less mature (3 of 8 survived) or immature (0 of 8 survivors) DCs. Nevertheless, intermediate-maturity DCs expressed substantial levels of Flt-3L, IGF-1, IL-1alpha and -1beta, IL-6, CCL-2, -3, -4, -9/10, -17, and -22, MIP-2, osteopontin, CCR-1, -2, -5, and -7, and CXCR-4. Taken together, our data clearly underscore the critical nature of employing DCs of full maturity for DC-based antitumor vaccination strategies.     

Proper processing of avian sarcoma/leukosis virus capsid proteins is required for infectivity

The formation of the mature carboxyl terminus of CA in avian sarcoma/leukemia virus is the result of a sequence of cleavage events at three PR sites that lie between CA and NC in the Gag polyprotein. The initial cleavage forms the amino terminus of the NC protein and releases an immature CA, named CA1, with a spacer peptide at its carboxyl terminus. Cleavage of either 9 or 12 amino acids from the carboxyl terminus creates two mature CA species, named CA2 and CA3, that can be detected in avian sarcoma/leukemia virus (R. B. Pepinsky, I. A. Papayannopoulos, E. P. Chow, N. K. Krishna, R. C. Craven, and V. M. Vogt, J. Virol. 69:6430-6438, 1995). To study the importance of each of the three CA proteins, we introduced amino acid substitutions into each CA cleavage junction and studied their effects on CA processing as well as virus assembly and infectivity. Preventing cleavage at any of the three sites produced noninfectious virus. In contrast, a mutant in which cleavage at site 1 was enhanced so that particles contained CA2 and CA3 but little detectable CA1 was infectious. These results support the idea that infectivity of the virus is closely linked to proper processing of the carboxyl terminus to form two mature CA proteins.     

Bioactive constituents from gum guggul (Commiphora wightii)

Bioactivity-directed fractionation and purification afforded cytotoxic components of Commiphora wightii. The exudates of C. wightii were extracted with EtOAc and the extract was subjected to repeated column chromatography. A fraction showing cytotoxic activity was characterized as a mixture of two ferulates with an unusual skeleton by spectral and chemical methods, including by NMR, GC-MS and chemical derivatization. This fraction also showed moderate scavenging effect against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals.